Yeast Cell Death (YCD) is a phenomenon in fungi that is possibly related to programmed cell death in animals. Recent studies show that common antifungal drugs activate a pro-YCD regulatory pathway but its effects are totally blocked by the simultaneous activation of an anti-YCD regulatory pathway. We have identified reactive oxygen species as a key component of the pro-YCD pathway and several components of the anti-YCD pathway. Natural compounds that specifically block one component of the anti-YCD pathway (such as Cyclosporin and FK506) greatly increase the potency of existing antifungal drugs by unmasking their fungicidal activity. Here we propose to develop assays of YCD suitable for high-through screening of chemical libraries to reveal new compounds that modulate pro- and anti-YCD factors. The assays under development must differentiate between dead cells and live non-proliferating cells and they must be applicable to diverse species of yeasts such as Saccharomyces cerevisiae and Candida albicans. The most direct assay will utilize high-throughput flow cytometry available at one MLSCN center whereas two other indirect assays will utilize instrumentation available at all MLSCN centers. We propose to develop all three assays for HTS format and evaluate the effectiveness of each using a variety of control strains and test conditions. Using the best assay and optimized conditions, a pilot screen will be performed to assess the frequencies of false and true positives in a broad chemical library. Secondary assays to rapidly identify and eliminate false positives will be developed for high-throughput application, if necessary. Finally, tertiary assays will be developed to rapidly map the point of action of each new chemical to the known factors in the anti-YCD pathway. These studies will greatly impact the final design and implementation of high-throughput screens for modulators of YCD. Such modulators will enable studies of YCD, programmed cell death, and lifespan control in fungi that are not genetically tractable. They will also provide new opportunities for control of fungal infections.